The current stable of licensed vaccines in the human and veterinary arenas is generally successful against what are termed “Class One pathogens.” Class One pathogens (such as poliovirus, smallpox, measles, mumps and rubella viruses) are those pathogens, which, in general: (1) infect or cause the most serious disease in children/young adults, (2) carry a relatively stable microbial genome, (3) have a natural history of disease which results in spontaneous recovery; and (4) induce durable memory, associated with polyclonal and multi-epitope antigen recognition.
In contrast, Class Two pathogens, such as, human rhinovirus (HRV), Foot-and-Mouth-Disease Virus (FMDV), viral influenza, HIV-1, malaria, tuberculosis, trypanosomes, schistosomes, leishmania, anaplasma, enterovirus, astrovirus, Norwalk viruses, toxigenic/pathogenic E. coli, Neisseria, Streptomyces, nontypeable haemophilus influenza, hepatitis C, cancer cells etc. are characterized by quite opposite features. For example, Class Two pathogens: (1) tend to infect and are transmitted in a significantly extended host age range, with infections occurring and reoccurring from childhood through the geriatric period; (2) exhibit microbial genetic instability in defined regions of their genome (a hallmark of the successful evolution of such pathogens); (3) in some cases, include spontaneous recovery of disease that frequently still leaves the host vulnerable to multiple repeated annual infections and/or the establishment of either a chronic/active or chronic/latent infectious state; (4) induce oligoclonal, early immune responses that are directed to a very limited set of immunodominant epitopes which provide either narrow strain-specific protection, no protection and/or enhanced infection; and (5) cause immune dysregulation following infection or vaccination, e.g. epitope blocking antibody, atypical primary immune response Ig subclasses, anamnestic cross reactive recall and inappropriate TH1 and/or TH2 cytokine metabolism.
At the immunologic level, infection with HRV may stimulate strain-specific immunity, but the host remains susceptible to re-infection by other serotypes of the virus. Characterization of immune responses against HRV suggests that the immune system recognizes and reacts to only a small number of immunodominant epitopes. Because the immunodominant epitopes are in highly variable sites that distinguish the various HRV serotypes, the immune response is highly strain-specific. Thus, an effective cross-protective vaccine against HRV must stimulate immune responses that are directed against more highly conserved regions of the virus, some of which may have previously been subdominant. In the case of HRV, a successful vaccine must overcome strain-specific immune responses to stimulate cross-protective immunity against 1) multiple serotypes and 2) evolving antigenic determinants.
Although some advances with regard to antigen delivery and expression have improved the immunogenicity of some Class Two microbial pathogens, current vaccine technologies have not readily translated into new, broadly effective and safe licensed vaccines for use in humans or animals. That may be due, in large part, to a poor understanding of the fundamental laws governing the vertebrate host defense system origin, repertoire development, maintenance, activation, senescence and co-evolution in similar and dissimilar environments.
Antigenic variation is an evolved mechanism to ensure rapid sequence variation of specific pathogen gene(s) encoding homologues of an individual protein antigen, usually involving multiple, related gene copies, resulting in a change in the structure of an antigen on the surface of the pathogen. Thus, the host immune system during infection or re-infection is less capable of recognizing the pathogen and must make new antibodies to recognize the changed antigens before the host can continue to combat the disease. As a result, the host cannot stay completely immune to the viral disease. That phenomenon stands as one of the more, if not, most formidable problem challenging modern vaccine development today.
Thus, it is not surprising that natural infection and vaccination fail to yield a more functional cross-reactive primary and anamnestic immunity as the repertoire development against those less immunogenic epitopes, which may be more conserved and capable of generating cross-strain immunity, are lower on the antigenic hierarchy.
The immunologic phenomenon whereby immunodominant epitopes misdirect the immune response away from more conserved and less immunogenic regions on an antigen was initially termed “clonal dominance”60 (Kohler et al., J Acquir Immune Defic Syndr 1992; 6:1158-68), which later was renamed as “Deceptive Imprinting” (Köhler et al., Immunol Today 1994 (10):475-8).
The immunologic mechanisms for immunodominance behind deceptive imprinting are not fully understood, and no one mechanism yet fully explains how or why certain epitopes have evolved to be immunoregulatory and immunodominant. The range of immune responses observed in the phenomena include: the induction of highly strain/isolate-specific neutralizing antibody capable of inducing passive protection in experimental animal model-viral challenge systems all the way to the induction of a binding non-protective/non-neutralizing, blocking and even pathogen-enhancing antibody that in some cases prevents the host immune system from recognizing the nearly adjacent epitopes, to interfering with CD4 T-cell help. The same decoying of the immune response through immunodominance resulting in a more narrowly focused set of epitopes is observed with T cells of the host in the development of helper and cytotoxic cell-mediated immunity. (Gzyl et al., Virology 2004; 318(2):493-506; Kiszka et al., J Virol. 2002 76(9):4222-32; and Goulder et al., J Virol. 2000; 74(12):5679-90).
Human Rhinoviruses (HRVs) are among the most common of human pathogens. It is estimated that each year the common cold is responsible for about 20 million missed work days, 22 million missed school days, and 27 million physician visits in the United States alone (Adams, Hendershot et al. 1999; Turner 2001; Mackay 2008). In addition, tens of billions of dollars per year are spent on prescription and over-the-counter medicines associated with treatments for the common cold (Bertino 2002). The estimated overall economic impact of colds in the U.S. in 2008 was nearly $40 billion a year composed of $17 billion from direct medical costs and $22.5 billion in indirect costs.
HRV is a highly contagious human pathogen that causes respiratory tract symptoms related to “the common cold” and exacerbates asthma and chronic pulmonary diseases. HRV is an unenveloped virus of the family Picornaviridae and is composed of 60 copies each of the viral capsid proteins VP-1, VP-2, VP-3, and VP4 and one copy of positive-sense RNA. The capsid proteins are translated in a genome-length polyprotein and cleaved to mature proteins by the viral protease-3C. The capsid proteins mediate binding to the cell receptor to facilitate virus entry and contain the primary virus neutralizing epitopes for immune targeting. HRV exists as a large number of serotypes dually classified based on 1) cell receptor usage and 2) antigenic relatedness. Viruses in the major group utilize the ICAM-1 receptor and those in the minor group use several members of the low density lipoprotein receptor that are almost ubiquitously expressed on many cell types. The serotypes are arranged with at least 3 clades (HRV-A, HRV-B, and HRV-C) based on genetic relationships.
To develop a strategy to overcome strain-specific immunity, it is necessary to understand the nature of native immune responses against HRV and other related members of the picornaviridae family. Within 2-3 weeks of infection or immunization with HRV virions, the immune system responds by developing humoral responses containing high titers of neutralizing antibody that are thought to help clear virus infections. However, the antibodies are directed against a small number of immune dominant epitopes that are located with genetically variable regions of the capsid proteins. Thus, infection or immunization with a virus or vaccine derived from one strain does not stimulate protective immunity against others. Because of the ubiquity of numerous HRV strains, vaccines that stimulate protection against one or a few serotypes are not effective.
Immune Refocusing Technology (IRT) was developed to over strain-specific immunity by reducing the antigenicity of immunodominant epitopes responsible for the strain-specific immune reactions. Using IRT, immunodominant epitopes are altered by site-specific mutagenesis to allow the immune system to develop responses to previously subdominant epitopes that participate in the development of more broadly protective immune responses.
FIG. 1 shows a diagram that described IRT using a model representing the trimeric influenza A/Aichi/68 hemagglutinin (HA) structure. In the molecule in Panel A, strain-specific antibodies (identified as naturally-occurring antibodies) are produced against highly variable immunodominant epitopes (shown as the blackened residues and identified with arrows). Using IRT, specific amino acid residues in the immunodominant epitopes are altered to reduce the antigenicity of the epitopes (depicted as light gray ovals). The rationally designed IIA molecule stimulates the production of antibodies to previously subdominant epitopes. The newly refocused antibodies (shown as novel cross-neutralizing antibodies) have enhanced cross-protective antiviral activities against heterologous viruses. In addition, the rationally designed antigen can be used to derive novel therapeutic antibodies with enhanced cross-protective properties.
The IRT can be applied to derive improved HRV vaccines that stimulate enhanced cross-protective immunity against multiple strains. Rationally designed immunogens can be engineered with mutations in the immunodominant epitopes such that the immune system responds against more broadly protective subdominant epitopes. The novel immunogens can be incorporated into whole virus particles or expressed as recombinant subunit antigens for vaccine production.